In Silico Docking Studies of Selected Flavonoids-Natural Healing Agents against Breast Cancer

Breast cancer is one of the most recurring worldwide diagnosed and deadliest cancers next to lung cancer with a high number of mortality rates among females (Benson and Jatoi, 2012). At global level, it accounted for more than 1.6 million new cases in 2010. The incidence or prevalence rate of the breast cancer in India is expected to be more than 90,000 in the coming years and over 50,000 women die each year (Parveen et al., 2013). A high level of Estrogen is linked with increased risk of breast cancer, which mediates its biological effects such as genesis, malignant progression, cell apoptosis and other important roles by binding to the Estrogen Receptor present in the breast cancer cells (Thomas and Gustafsson, 2011; Fatemeh et al., 2012). The Estrogen Receptor mainly exists in two forms: Estrogen Receptor alpha and Estrogen Receptor Beta. Estrogen receptors alpha (ER-α) is mainly expressed in the uterus, vagina, mammary gland, liver, pituitary gland (Waraphan et al., 2011). The major causes of breast cancer are identified by abnormal expression of Estrogen Receptor α-positive affecting about 70% of the primary breast cancer patients (Dickson and Stancel, 2000; Fuqua, 2001; Ariazi et al., 2006; Chao-Yang et al., 2013; Kumaraswamy and Suneetha, 2013). The Estrogen Receptor alpha plays a pivotal role in controlling transcription of nuclear DNA necessary for mammary


Introduction
Breast cancer is one of the most recurring worldwide diagnosed and deadliest cancers next to lung cancer with a high number of mortality rates among females (Benson and Jatoi, 2012).At global level, it accounted for more than 1.6 million new cases in 2010.The incidence or prevalence rate of the breast cancer in India is expected to be more than 90,000 in the coming years and over 50,000 women die each year (Parveen et al., 2013).

In Silico Docking Studies of Selected Flavonoids -Natural Healing Agents against Breast Cancer
Jeyabaskar Suganya, Mahendran Radha*, Devi Leimarembi Naorem, Marimuthu Nishandhini gland development (Hayashi et al., 2003) and it is also an essential factor for breast cancer signaling network (Pantea et al., 2012).It also regulates cell proliferation and differentiation through a paracrine mechanism (Hong et al., 2013).Hence, the inhibition of Estrogen Receptor has become a major approach for preventing and treating breast cancer (Salih and Fentiman, 2001).
The drugs which are currently used, for the treatments of breast cancer were Tamoxifen, Raloxifene, Toremifene.(Fabian and Kimler, 2001).Ingestion of this drug was based on interfering with either estrogen production or estrogen action which causes so many side effects such as blood clots, strokes, uterine cancer, or cataracts (Parkkari et al., 2003;Mojgan et al., 2009;Andrew et al., 2011).The side effects of these drugs make the need for the necessity of new improved drugs.
The side effects of the currently used drug made us to explore an alternative and traditional approach to finding out new drug compound from the natural Flavonoid, compounds which are having anti breast cancer activity and also not having any side effects to human normal cell (Kawaii et al., 1999;Pouget et al., 2001).These plant compounds which are also having high binding affinity for breast cancer receptors could lead to breast cancer treatment by docking method and to determine the druglikeness of these new molecules by estimation of the Lipinski's Rule of Five.
Flavonoids functions as selective Estrogen Receptor modulators (SERMs) (Pritchard, 2001;Park and Jordan, 2002) changes the endogenous activities of Estrogen Receptor, which will slow down or prevent the developments of breast cancers (Limer and Speirs, 2004;Jarzabek et al., 2009).Flavonoids are large group of natural polyphenolic compounds present in fruits, vegetables, cereals, tea, red wine, legumes and exhibit antioxidant, anticarcinogenic, anti-inflammatory, antiproliferative, antiangiogenic, and antiestrogenic (or estrogenic) properties (Shuzhong et al., 2004;Paola Galluzzo and Maria, 2006;Miller and Snyder, 2012;Sanaz et al., 2012;Stapel et al., 2013).They also play a fundamental biological role in the regulation of breast cancer cell line proliferation.Several Epidemiological and in vivo studies also recommended that a high intake of Flavonoids may be associated with a reduced risk of cancer (Chang et al., 2013).Flavonoids can be further divided, according to chemical structure, into different groups.Flavonoids listed in the (Table 1), are already reported in various literatures that they possess anti breast cancer activities (Shi et al., 2011;Feng et al., 2013;Priya et al., 2013).

Protein preparation
The three dimensional crystal structure of Human Estrogen Receptor with PDB: 2IOG (Dykstra KD et al., 2007) ACETAMIDE was retrieved from the Protein Data Bank (http://www.rscb.org/pdb)(Berman HM et al., 2002).The complexes bound to the receptor molecule, all the heteroatoms and the non-essential water molecules were removed and finally hydrogen atoms were merged to the target receptor molecule using Argus Lab.

Ligand preparation
Totally 19 flavonoids were identified from the Pubmed literatures which shows inhibitory effects towards breast cancer.The three dimensional structure of the flavonoids was downloaded in sdf format using Pubchem and converted to PDB format using Pymol and further used for docking studies.

Active site identification of human estrogen receptor
The catalytic sites of human Estrogen Receptor along with area and volume of binding pocket was carried out with Computed Atlas of Surface Topography of Proteins (Castp) program (http://cast.engr.uic.edu)(Binkowski et al., 2003).

Docking studies using argus lab
The docking analysis of Human Estrogen Receptor with 19 Flavonoids was carried out by Argus lab docking software which is most commonly available software.All the parameters used in Argus lab docking were selected by default.Calculation type was set to "dock" mode and "flexible mode" was selected for the ligand.Grid resolution was set to 0.40 Å. Least energy indicated the easy binding character of ligand and receptor (Oda et al., 2007).

Lipinski rule of five of the ligand molecule
Using the Molinspiration server (http://www.molinspiration.com/)Molecular properties and drug likeness of the compounds was examined on the basis of "Lipinski's Rule of Five" (Lipinski et al., 2001).The Lipinski's rule, formulated by Christopher A Lipinski in 1997 is a rule of thumb to evaluate drug likeness which states that an orally active drug has no more than one violation of following criteria i,e., has not more than 5 hydrogen bond donors, not more than 10 hydrogen bond acceptors, molecular weight below 500 Daltons, partition co-efficient log P less than 5.

Structure of the target protein
Human Estrogen Receptor (2IOG) has been exploited as a main therapeutic target for Breast Cancer.The three dimensional structure of Human Estrogen Receptor retrieved from the Protein Data Bank with PDB ID: 2IOG determined by X-Ray crystallography at a resolution of 1.60 (Å) was visualized in Pymol.It contains 246 amino acids and has been shown in the Figure 1.

Docking analysis
The predicted active residues were used as the catalytic sites for nineteen natural Flavonoid compounds used for docking studies.The results of the interaction between the active site residues of target Human Estrogen Receptor protein and 19 Flavonoid compounds were shown in the Table 2.By analyzing the docking interactions, Chrysin and Equol were found to have the highest activation energy of -11.0189 kcal/mol (Figure 3) and -10.8354 kcal/mol (Figure 4) when compared with other compounds which are having the activation energy of < -10.5 kcal/mol.Thus the docking results were analyzed and finally reported that among the 19 plant compounds, Chrysin and Equol exhibits the best binding interaction with the human Estrogen Receptor and further it could be useful for identification and development of new preventive and therapeutic drug against breast cancer.

Evaluation of drug likeness
Further the 19 Flavonoid Compounds were checked for Lipinski's Rule of Five using Molinspiration software tool.The results of molecular properties and Drug likeness of the Flavonoids were shown in the Table 3 and  Table 4.By evaluating the drug likeliness we conclude that all the nineteen anti breast cancer compounds have passed through the Lipinski rule's of five criteria.Three compounds namely Epigallocatechin gallate, Myricetin and Gallocatechol have acquired more than 10 hydrogen bond acceptor and more than 5 Hydrogen bond donor have failed to satisfy the Lipinski rule's of five and hence cannot be used as an oral drug.Finally two compounds Chrysin and Equol satisfies all the properties of pharmacological or biological activity with a best result when compared with other compounds .Thus we conclude that the above two compounds would be more effective as an active drug for human consumption.
The development of novel compounds with biological activity is an urgent need for developing effective drugs.In the present study, docking results revealed the binding interactions between the Human Estrogen Receptor protein and the 19 natural Flavonoid compounds.Among those compounds, Chrysin and Equol were found to have the best binding energy of greater than 10.5 kcal/mol and also satisfies the Lipinski Rule's of five which provide the strong recommendation for the compounds to be used as an oral drug.On the whole, we conclude that Flavonoids Crysin and Equol could be potent anti-breast cancer drugs against Human Estrogen Receptor which may be worth for further investigations to enrich the activity of the natural Flavonoid compounds especially in experimental studies with clinical trials to determine the dosage of safety levels.